Campylobacter sputorum subsp mucosalis and Campylobacter hyointestinalis infections in the intestine of gnotobiotic pigs

At 4 days of age, 7 gnotobiotic pigs were orally inoculated with broth cultures of both Campylobacter sputorum subsp mucosalis (CSM) and Campylobacter hyointestinalis (CH). One pig was killed and evaluated each week for 7 weeks. Forty-eight hours after inoculation, CH and CSM were recovered from the feces of the pigs; thereafter, only CH was recovered. Organisms morphologically typical of Campylobacter sp were observed on the mucosal surface and on the crypt epithelial cells of the ileum, cecum, and colon from post-inoculation weeks (PIW) 2 through 7. Bacteria were clustered around the surface opening of goblet cells in pigs at PIW 6 and 7. Crypt epithelial cell proliferation and intracellular bacteria were not seen, except in 1 pig (killed at PIW 7) in which intracellular bacteria were seen only in the cecum. Therefore, CSM and CH did not induce porcine proliferative enteritis in gnotobiotic pigs.     

The isolation of Campylobacter hyointestinalis from a pig in South Africa

The isolation of a strain of Campylobacter hyointestinalis from a piglet is described. The animal originated from a farm where another animal showed signs of intestinal adenomatosis. The animal from which the isolation was made had diarrhoea, and an enteropathogenic Escherichia coli was also isolated. No pathological changes indicative of intestinal adenomatosis were detected in this animal.     

Infection of established cell lines with Campylobacter mucosalis

Campylobacter mucosalis (CM) and other Campylobacter spp. mainly from cases of the porcine proliferative enteropathies were used to infect cell line cultures. CM attached to and persisted intracellularly in PK (15), MDBK, BHK, HeLa, INT-407 and other cells; in PK (15) this infection resulted in a marked cytopathic effect and total destruction of the cell culture in 10 days. MDBK, INT-407, BHK and HeLa cultures were more resistant to the effects of infection. CM did not adhere to Vero, LLCMK₂ or CEF cells and no cytopathic changes took place. The other Campylobacter spp tested did not attach to cell cultures and cell death occurred within 4 days. Attempts to passage CM were unsuccessful due to a decrease in the number of bacteria at each passage.     

Behaviour of Campylobacter sputorum subspecies mucosalis in gnotobiotic pigs

Gnotobiotic pigs were dosed orally with Campylobacter sputorum subspecies mucosalis, either alone, or combined with rotavirus or non-pathogenic Escherichia coli and Streptococcus bovis to study the behaviour of C s mucosalis in defined conditions, to assess intracellular parasitism of enterocytes by C s mucosalis, and if possible to establish an experimental model of porcine intestinal adenomatosis. C s mucosalis colonised the gut of gnotobiotic pigs, persisting for up to 47 days after infection, but did not induce adenomatosis. Despite evidence of limited penetration of the mucosa up to two days after infection, the majority of C s mucosalis remained in the gut lumen. Rotavirus did not enhance invasion of enterocytes by C s mucosalis. The presence of E coli and S bovis caused an increase in the total numbers of C s mucosalis in the gut, but did not affect their distribution. Thus C s mucosalis was largely non-pathogenic in gnotobiotic pigs.     

The first isolations of Campylobacter mucosalis from pigs in South Africa

The first isolations of Campylobacter mucosalis in South Africa are described. Isolations were made from a 6-week-old weaner pig with necrotic enteritis and from 2 gingival swabs of suckling piglets from herds with histories of porcine intestinal adenomatosis. The isolates were serologically identified as being serotype A strains.     

Southern blot analysis of strain variation in Campylobacter mucosalis.

A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.     

Comparison of selective media for the isolation of Rhodococcus equi and description of a new selective plating medium

In order to improve the isolation rate of Rhodococcus equi from animals and soil, the efficacy of four previously described selective media (CAZ-NB, M3T, NANAT and TINSDALE) and that of four other media (NC, PNP, TCP and TVP) composed by us was compared and evaluated. Two selective plating media proved to be the best for the isolation of R. equi from contaminated samples. One of them was CAZ-NB containing ceftazidime, novobiocin and cycloheximide, while the other was the newly composed TCP containing trimethoprim, cefoperazone, polymyxin B, cycloheximide and potassium tellurite as selective components. These two media allowed the growth of at least 62-72% of R. equi present in the artificially contaminated samples, and the inhibition of unwanted contaminant bacteria and fungi was satisfactory with both media. TCP medium proved to be superior to CAZ-NB since the colony morphology of R. equi was much more characteristic (shiny, smooth, black colonies 3-5 mm in diameter) on it, and it inhibited the unwanted contaminant bacterial and fungal flora more effectively, especially in the case of faecal and soil samples. Therefore, TCP is recommended as a new, highly selective plating medium for the isolation of R. equi from contaminated samples.     

Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens

A total of 2450 samples of feces, intestinal contents and colon mucosal scrapings were bacteriologically examined. A total of 53 strains of Treponema sp. were isolated, and 45 strains of Bacteroides sp., 30 strains of E. coli, 30 strains of Micrococcus sp. and 10 strains of Streptococcus D isolates were randomly selected. Growth promoting studies showed statistically significant stimulation of Treponema sp. growth by yeast extract, chicken egg yolk and rumen fluid. Different growth inhibitors were also tested. For selective medium the following inhibitors were selected: spectinomycin, colistin, vancomycin, brilliant green. Optimal concentrations of these inhibitors in the medium were determined. Finally TSA medium supplemented with 0.05% yeast extract, 5% bovine blood, 0.01% DTT, 400 micrograms spectinomycin, and 250 micrograms/ml vancomycin, appeared to be optimal selective medium for intestinal Treponema sp. isolation. Quantitative studies showed that the number of Treponema C.F.U. on Songers et al. medium with spectinomycin and on spectinomycin-vancomycin medium, did not differ significantly. The number of overgrowing bacteria was statistically significantly lower on spectinomycin-vancomycin medium, than Songers et al. selective medium with spectinomycin. The TSA supplemented with blood, yeast extract 50 micrograms/ml of colistin and 1 microgram/ml of brilliant green was less selective than spectinomycin-vancomycin medium and inhibited some strains of Treponema sp. In the case of spectinomycin-vancomycin resistant of overgrowing bacteria, colistin-brilliant green medium may be suitable for isolation of Treponema sp.     

Comparison of two selective media for the recovery, isolation, enumeration and differentiation of Rhodococcus equi

The use of selective media to facilitate the isolation of Rhodococcus equi from environmental and clinical samples has aided studies of the ecology of R. equi and the epidemiology of disease caused by R. equi. Here, we compared the efficacy of two selective media (NANAT and modified CAZ-NB) for the recovery of six defined strains of R. equi and for the isolation and enumeration of both avirulent and virulent R. equi from 60 paired soil samples from horse farms using colony blotting and DNA hybridisation. No difference was found between the two media in the recoverability of defined strains of R. equi or the proportion of soil cultures positive for R. equi or virulent R. equi. NANAT medium was significantly less inhibitory of bacterial growth from soil culture compared to mCAZ-NB (P = 0.001), but there was no difference between the media in the number of R. equi colonies recovered. Soil cultured on mCAZ-NB medium yielded a significantly greater number of virulent R. equi colonies than NANAT (P = 0.03). The proportion of R. equi that were virulent in soil cultures on mCAZ-NB (32%) was more than three times that seen in cultures on NANAT (9%). Thus modified CAZ-NB appeared to be a better selective media for studies where the optimal recovery of virulent R. equi is required, such as in studies of the gastrointestinal carriage of virulent R. equi and of subclinically infected foals.     

Comparative studies on enrichment and selective media for isolation of Salmonellae from faecal specimens.

Enrichment media (tetrathionate, selenite and Rapp ap ort broths) and selective media (desoxycholate citrate agar and brilliant green agar) were tested in different combinations to ascertain their capacity for isolation of salmonella bacteria. The material consisted of 299 samples of cattle faeces from two herds infected with salmonella (Table 1), and of 111 artificially contaminated samples of pig faeces (Table 3). The tetrathionate and selenite broths were equally useful for the material as a whole, whereas the results varied between different species of salmonella which is of great practical interest. The number of salmonella isolations was much lower when enrichment with Rappaport broth was used. The rate of salmonella isolations can often be increased by parallel enrichments with two different media. Of the selective agar media tested, brilliant green agar was superior to desoxycholate citrate agar.     

Experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis with special reference to the oral cavity

After oral exposure Campylobacter sputorum subsp. mucosalis became established in the mouth of piglets for up to eight weeks. C. sputorum subsp. mucosalis was not isolated from the same pigs each week, although some pigs were positive for a number of consecutive weeks. Spread also took place from orally dosed piglets to an undosed litter mate maintained with them. Other catalase negative, serologically distinct Campylobacters were also demonstrated in the oral cavity.